The Role of RNA Structure in the Hepatitis B Virus Lifecycle

RNA 结构在乙型肝炎病毒生命周期中的作用

• 批准号: 10117730
• 负责人: Michael J Bouchard
• 金额: $ 22.23万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-11 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10117730
• 关键词: Acylation; Adopted; Affect; Binding; Biochemical; Bioinformatics; Biological Assay; Biological Products; Boston; Capsid; Capsid Proteins; Cell Culture Techniques; Cell Nucleus; Cells; Chemicals; Chronic Hepatitis B; Circular DNA; Cytosol; DNA; DNA Viruses; Development; Drug Targeting; Elements; Environment; Episome; Future; Genome; Goals; Hepatitis B Virus; Hepatocyte; Hydroxyl Radical; Immunomodulators; Infection; Length; Link; Lipids; Maps; Messenger RNA; Methods; Mutation; Mutation Analysis; Nucleocapsid; Open Reading Frames; Polymerase; Primary carcinoma of the liver cells; Primer Extension; Proteins; RNA; RNA Viruses; RNA-Directed DNA Polymerase; Reverse Transcription; Role; Structure; Techniques; Therapeutic; Transcript; Translating; Translation Initiation; Translations; Untranslated RNA; Vaccines; Viral; Viral Genome; Viral Packaging; Virus; Virus Replication; analog; anti-hepatitis B; dimethyl sulfate; drug development; ds-DNA; high reward; next generation sequencing; novel; novel strategies; pgRNA; small molecule; stem; viral DNA; viral RNA; viral rebound

SUMMARY Globally, a chronic hepatitis B virus (HBV) infection is the most common cause of hepatocellular carci- noma (HCC). An estimated 240 million people are chronically HBV-infected worldwide, and approved treatments are rarely curative. HBV has a DNA genome enclosed in a virally encoded protein capsid surrounded by a protein/lipid envelope. After infection of a hepatocyte, the viral genome is delivered to the nucleus and converted to an episome referred to as covalently closed circular (ccc)DNA. cccDNA is not targeted by current anti-HBV therapies, and its stability is linked to viral rebound when these therapies are stopped. Attempts to target cccDNA have not succeeded, and there is a need for new anti-HBV therapies. cccDNA is not directly replicated but is the template for HBV RNA transcripts. One of the transcripts, the pregenomic (pg)RNA, is encapsidated and reverse- transcribed by the virally encoded reverse-transcriptase/polymerase (RT/Pol) to generate the HBV DNA genome. Recent advances in RNA structure probing techniques have led to the discovery of RNA structural elements in several viruses. Many of these elements have critical roles in the viral lifecycle, and targeting RNA structures in viral genomes could open new avenues of drug development. Despite the therapeutic potential, the role of RNA structure in the HBV lifecycle is largely unexplored. The multi-functional HBV pgRNA is the template for reverse transcription and is also translated to produce the core (capsid) and RT/Pol proteins. The RT/Pol open-reading frame (ORF) is 3’ of the core ORF on pgRNA and overlaps with the 3’ end of the core ORF; synthesis of RT/Pol requires internal initiation of translation by a mechanism that is not understood. RNA structures referred to as epsilon (ε) stem-loops are located near the 5’- and 3’-ends of pgRNA, yet only the 5’ ε is required for pgRNA encapsidation. Due to limitations of available techniques, prior studies focused on local regions in HBV RNA transcripts, and many questions remain. For example, whether encapsidated and translated pgRNAs are structurally different, how internal initiation of RT/Pol synthesis is regulated, and why only the 5’ ε affects pgRNA encapsidation are incompletely understood or entirely unknown. New techniques to examine RNA structure in living cells, such as SHAPE-MaP (selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling) and DMS-MaP (dimethyl sulfate probing and mutational profiling), couple RNA chemical probing techniques with next-generation sequencing to identify RNA structures over the entire length of an RNA transcript. We will use these techniques to define RNA structural elements in HBV pgRNA and the role of these elements in the HBV lifecycle. We hypothesize that pgRNA harbors structural elements that have critical roles in the HBV lifecycle, and our studies could identify novel and highly specific targets for blocking HBV replication and the development of HCC.

概括 在全球范围内，慢性乙型肝炎病毒（HBV）感染是肝细胞癌的最常见原因。 坏疽性口炎（HCC）。据估计，全球有 2.4 亿人长期感染 HBV，并且批准的治疗方法 很少有疗效。 HBV 的 DNA 基因组封闭在病毒编码的蛋白质衣壳中，衣壳周围有 蛋白质/脂质包膜。感染肝细胞后，病毒基因组被递送至细胞核并转化 附加体称为共价闭合环状 (ccc)DNA。 cccDNA 不是当前抗 HBV 药物的靶标 疗法，其稳定性与这些疗法停止后病毒反弹有关。尝试针对 cccDNA 尚未成功，因此需要新的抗乙型肝炎疗法。 cccDNA 不是直接复制的，而是 HBV RNA 转录本的模板。其中一种转录物，即前基因组 (pg)RNA，被衣壳化并反向- 由病毒编码的逆转录酶/聚合酶 (RT/Pol) 转录，生成 HBV DNA 基因组。 RNA结构探测技术的最新进展导致RNA结构的发现 多种病毒中的元素。其中许多元素在病毒生命周期中发挥着关键作用，并且靶向 RNA 病毒基因组的结构可以开辟药物开发的新途径。尽管具有治疗潜力， RNA 结构在 HBV 生命周期中的作用很大程度上尚未被探索。以多功能HBV pgRNA为模板 用于逆转录，也可翻译产生核心（衣壳）和 RT/Pol 蛋白。 RT/Pol 开放阅读框（ORF）位于pgRNA上核心ORF的3'端，并与核心ORF的3'端重叠； RT/Pol 的合成需要通过一种尚不清楚的机制进行内部启动。核糖核酸 被称为 epsilon (ε) 茎环的结构位于 pgRNA 的 5' 端和 3' 端附近，但只有 5' ε 是 pgRNA 衣壳化所必需的。由于现有技术的限制，先前的研究主要集中在局部 HBV RNA 转录本中的区域，但仍然存在许多问题。例如，是否封装和翻译 pgRNA 在结构上不同，如何调节 RT/Pol 合成的内部启动，以及为什么只有 5' ε 影响 pgRNA 衣壳化的因素尚未完全了解或完全未知。 检查活细胞中 RNA 结构的新技术，例如 SHAPE-MaP（选择性 2′-羟基 通过引物延伸和突变分析进行酰化分析）和 DMS-MaP（硫酸二甲酯探测和 突变分析），将 RNA 化学探测技术与新一代测序相结合来识别 RNA RNA转录本全长的结构。我们将使用这些技术来定义 RNA 结构 HBV pgRNA 中的元素以及这些元素在 HBV 生命周期中的作用。我们假设 pgRNA 含有在 HBV 生命周期中起关键作用的结构元件，我们的研究可以识别新的和 阻断 HBV 复制和 HCC 发展的高度特异性靶标。